The mechanism(s) by which mammalian cells produce N-fatty acylglycines (NAGs, R-CO-NH-CH2-COOH, where R is a fatty acid) and the other lipoamino acids is poorly understood. The NAGs, the lipoamino acids, and the primary fatty acid amides (PFAMs, R-CO-NH2) are members of a larger class of fatty acid amide cell signaling lipids, the fatty acid amides. The best understood members of the fatty acid amide family are the N-acylethanolamines (NAE, R-CO-NH-CH2-CH2-COOH). One NAE of considerable importance is N-arachidonoylethanolamine, also known as ananadamide, the endogenous cannabinoid ligand found in the mammalian brain. The NAGs and the other lipoamino acids are underappreciated members of the fatty acid amide family, but accumulating evidence demonstrates that these are also important in human health. Work carried out in our laboratory and by others suggests that the NAEs, NAGs, and PFAMs are tied together in a precursor-product relationship: NAEs -> N-fatty acylglycines -> PFAMs. The NAGs are also produced by a reaction between the fatty acyl-CoA thioester and glycine: acyl-CoA + glycine -> NAG + CoA-SH. This exciting sequence indicates that the roles served by one fatty acid amide can be radically altered by metabolic conversion to another fatty acid amide. The enzymes catalyzing these reactions could then play important regulatory functions simply by interconverting the fatty acid amides. The focus of this application is to provide fundamental information regarding the biosynthesis of the NAGs and other lipoamino acids and to firmly establish metabolic links between members of the fatty acid amide family. We specifically propose to: (a) characterize a novel set of mammalian glycine N-acyltransferases (GLYATs) and define the role played by these enzymes in NAG biosynthesis and (b) determine the relative contributions to total NAG production made from the two precursors, the acyl-CoAs and the NAEs. Key to our research efforts are two model cell lines, mouse neuroblastoma N18TG2 cells and sheep choroid plexus, cells that are known to produce the NAEs, the NAGs, and the PFAMs. PUBLIC HEALTH RELEVANCE: The proposed research represents an interdisciplinary effort to understand the pathways for the biosynthesis and metabolism of the N-fatty acylglycines (NAGs, R-CO-NH-CH2-COOH). Over the past 20 years, it has become increasingly clear that the fatty acid amides are a broad family of cell-signaling lipids tremendously important to human health. The members of the fatty acid amide family include the N-acylethanolamines (NAEs), the lipoamino acids, the N-acyldopamines (NDAs), and the primary fatty acid amides (PFAMs). The N-fatty acylglycines represent a subset of the known mammalian lipoamino acids. Recent studies have suggested that members of the fatty acid amide family are metabolically linked as follows: NAEs -> NAGs -> PFAMs. Our goal is to better define the metabolic relationships between the members of the fatty acid amide family and elaborate the enzymes catalyzing these conversions. A full understanding of NAG metabolism and the enzymes involved is essential to ultimately developing new information valuable in the development of novel therapeutic strategies to treat chronic pain.